Process for enhancing the viable counts of lactic acid bacteria and useful compositions thereof

ABSTRACT

Disclosed herein is a composition containing turmeric starch for use as a prebiotic plant fiber. Also disclosed is a method to increase the viable counts of  Bacillus coagulans  MTCC 5856 by co-culturing with turmeric starch and the production of short chain fatty acids (SCFA) by  Bacillus coagulans  MTCC 5856 using turmeric starch.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/622,616 filed on Jun. 14, 2017, which is a continuation of U.S.patent application Ser. No. 14/839,923, filed on Aug. 29, 2015, which inturn claims the benefit of priority to U.S. Provisional Application No.62/043,599, filed on Aug. 29, 2014, and 62/063,453, filed on Oct. 14,2014. The disclosure of the prior applications is hereby incorporated byreference herein in its entirety.

BACKGROUND Field of the Invention

The invention in general relates to Bacillus coagulans (Lactic acidbacteria). More specifically, the invention relates to (i) the growthpromotional activity of natural plant based fibers on Bacillus coagulansMTCC 5856; (ii) The production of short chain fatty acids (SCFA) byBacillus coagulans MTCC 5856 using plant based natural fibers; and (iii)the combination of natural plant based fibers and Bacillus coagulansMTCC 5856 to inhibit Gram Negative pathogenic bacteria.

Description of Prior Art

Combining multistrain probiotics (probiotic bacteria) and prebiotics toachieve enhanced immunosupportive effects is well known in the art.Specifically combining probiotics with natural plant based fibers toformulate synbiotics is reported as a promising therapeutic approach(Stig Bengmark and Robert Martindale. “Prebiotics and Synbiotics inClinical Medicine”. Nutr Clin Pract vol 20 244-261, April 2005). Thesuccess of such an approach depends on carefully choosing specificprobiotic microorganisms whose viable count is effectively enhanced bynatural plant based fibers that are resistant to both enzymatic and acidhydrolysis in the gut. These studies are critically important toaccommodate the performance of host animals exposed to symbiotic dietregimes given the teaching that there are limitations to fiber digestionand utilisation by microbes in terms of microbial accessibility tosubstrates, physical and chemical nature of fibers (forage) and alsokinetics of the digestive process (Gabriella A. Varga and Eric S.Kolver, “Microbial and Animal Limitations to Fiber Digestion andUtilization”, J. Nutr. May 1, 1997 vol. 127 no. 5 819S-823S).

It is the principle objective of the present invention to evaluate theperformance of selected natural fibers (enzyme and acid hydrolysisresistant) to enhance the viable counts of Bacillus coagulans MTCC 5856.

It is also another objective of the present invention to evaluate theability of the synbiotic composition (natural fibers and Bacilluscoagulans MTCC 5856) to inhibit pathogenic Gram negative bacteria.

It is yet another objective of the present invention to evaluate theability of the synbiotic composition (natural fibers and Bacilluscoagulans MTCC 5856) to produce desired short chain fatty acids, saidproperty having profound therapeutic applications.

The present invention fulfils the aforesaid objectives and providesfurther related advantages.

Deposit of Biological Material

The deposit of biological material Bacillus coagulans bearing accessionnumber MTCC 5856, mentioned in the instant application has been made on19 Sep. 2013 at Microbial Type Culture Collection & Gene Bank (MTCC),CSIR-Institute of Microbial Technology, Sector 39-A. Chandigarh—160036,India.

SUMMARY

Disclosed is (i) the growth promotional activity of natural plant basedfibers on Bacillus coagulans MTCC 5856; (ii) the combination of naturalplant based fibers and Bacillus coagulans MTCC 5856 to inhibit Gramnegative pathogenic bacteria and (iii) the production of short chainfatty acids (SCFA) by Bacillus coagulans MTCC 5856 using plant basednatural fibers.

Other features and advantages of the present invention will becomeapparent from the following more detailed description, taken inconjunction with the accompanying images, which illustrate, by way ofexample, the principle of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and IC show the graphical representation of the increase inviable colony count of Bacillus coagulans MTCC 5856 in presence ofdifferent natural plant fibers alone (%, w/v).

FIGS. 2A, 2B and 2C show the graphical representation of the increase inviable colony count of Bacillus coagulans MTCC 5856 in presence ofdifferent natural plant fibers (%, w/v) in MRS media (devoid ofdextrose) (0.5, 1.0, 2.0%, w/v).

FIG. 3 is the graphical representation of inhibition of E. coli ATCC25922 growth by B. coagulans MTCC 5856 when co-cultured in plant basednatural fibers as media. Average mean of viable counts are expressed inlog₁₀ cfu/ml.

FIG. 4 shows the production of total short chain fatty acid (acetate,butyrate, and propionate) by the B. coagulans MTCC 5856 from Fenugreekseed fibers (FSF), Lycium barbarum seed fibers (LSF), Flax seed fibers(FLSF), Coconut Fibers (CF), Ginger rhizome fibers (GRF), Amla FruitFiber (Soluble+Insoluble) (AFF), Amla Soluble Fibers (ASF), AmlaInsoluble Fibers (AIF), Psyllium husk Fibers (PHF), Cranberry seedFibers (CSF), Fructooligosaccharide (FOS), MRS Media (MRS), and MRSMedia devoid of Dextrose (MRSD). Average mean of the SCFAs are expressedin mg per gram of fiber.

FIG. 5 shows the graphical representation of the acid hydrolysis ofturmeric starch.

FIG. 6 shows the graphical representation of the Porcine pancreaticenzymatic hydrolysis of turmeric starch.

FIG. 7 show the graphical representation of the increase in viablecolony count of Bacillus coagulans MTCC 5856 in presence of turmericstarch (%, w/v) in MRS media (devoid of dextrose) (0.5, 1.0, 2.0%, w/v).

DETAILED DESCRIPTION

In the most preferred embodiment, the present invention relates to amethod of increasing the viable colony count of Bacillus coagulans MTCC5856 said method comprising step of growing Bacillus coagulant MTCC 5856in the presence of natural plant fibers selected from the groupconsisting of Trigonella foenum-graecum (fenugreek) seed fibers, Lyciumbarbarum seed fibers, Linum usilatissimum (Flax) seed fibers, Cocosnucifera (Coconut) fibers, Zingiber officinale (Ginger) rhizome fibers,Emblica officinals (Amla) fruit fibers, Plantago ovata (Psyllium) fibersand Vaccinium oxycoccos (Cranberry) seed fibers.

In another most preferred embodiment, the present invention relates to amethod of inhibiting pathogenic Gram negative bacteria said methodcomprising step of bringing to contact said Gram negative bacteria withBacillus coagulans MTCC 5856 co-cultured with natural plant fibersselected from the group consisting of Trigonella foenum-graecum(fenugreek) seed fibers, Lycium barbarum seed fibers, Linumusitatissimum (Flax) seed fibers, Cocos nucifera (Coconut) fibers,Zingiber officinale (Ginger) rhizome fibers, Emblica officinalis (Amla)fruit fibers, Plantago ovata (Psyllium) fibers and Vaccinium oxycoccos(Cranberry) seed fibers.

In yet another most preferred embodiment, the present invention alsorelates to a method of producing short chain fatty acids by co-culturingBacillus coagulans MTCC 5856 with natural plant fibers selected from thegroup consisting of Trigonella foenum-graecum (fenugreek) seed fibers,Lycium barbarum seed fibers, Linum usitatissimum (Flax) seed fibers,Cocos nucifera (Coconut) fibers, Zingiber officinale (Ginger) rhizomefibers, Emblica officinalis (Amla) fruit fibers, Plantago ovata(Psyllium) fibers and Vaccinium oxycoccos (Cranberry) seed fibers. Inalternate embodiments, the present invention also relates to a method ofprotecting against diet-induced obesity and insulin resistance in themammalian gut by administering composition comprising Bacillus coagulansMTCC 5856 with natural plant fibers selected from the group consistingof Trigonella foenum-graecum (fenugreek) seed fibers, Lycium barbarumseed fibers, Linum usitatissimum (Flax) seed fibers, Cocos nucifera(Coconut) fibers, Zingiber officinale (Ginger) rhizome fibers, Emblicaofficinalis (Amla) fruit fibers, Plantago ovata (Psyllium) fibers andVaccinium oxycoccos (Cranberry) seed fibers to bring about the effect ofprotection against diet induced obesity and insulin resistance.

In another most preferred embodiment, the invention discloses acomposition containing turmeric starch for use as a prebiotic plantfiber. In a related embodiment, the turmeric starch is isolated from thespent rhizomes of Curcuma longa.

In an another preferred embodiment, the invention discloses a methodcomprising co-culturing Bacillus coagulans with turmeric starch fibres,wherein the viable colony count of Bacillus coagulans is increased bythe co-culturing method.

In an another preferred embodiment, the invention discloses a method ofproducing short chain fatty acids, comprising performing the method ofco-culturing Bacillus coagulans with turmeric starch fibres underfermenting conditions to produce short chain fatty acids. In a relatedembodiment, the short chain fatty acids are one or more selected fromthe group consisting of acetic acid, propionic acid, and butyric acid.In another related embodiment, the Bacillus coagulans and the turmericstarch are fermented under anaerobic conditions.

In a preferred embodiment, the Bacillus coagulans is Bacillus coagulansMTCC 5856.

In an another preferred embodiment, the invention discloses a methodcomprising co-culturing Bacillus coagulans MTCC 5856 with turmericstarch fibres, wherein the viable colony count of Bacillus coagulansMTCC 5856 is increased by the co-culturing method.

In an another preferred embodiment, the invention discloses a method ofproducing short chain fatty acids, comprising performing the method ofco-culturing Bacillus coagulans MTCC 5856 with turmeric starch fibresunder fermenting conditions to produce short chain fatty acids. In arelated embodiment, the short chain fatty acids are one or more selectedfrom the group consisting of acetic acid, propionic acid, and butyricacid. In another related embodiment, the Bacillus coagulans MTCC 5856and the turmeric starch are fermented under anaerobic conditions.

The specific examples included herein below illustrate the aforesaidmost preferred embodiments of the present invention.

Example I

Method of Preparing Fibers

Trigonella Foenum-Graecum (Fenugreek) Seed Fibers

Trigonella foenum-graecum (also known as Fenugreek) seeds were collectedfrom local market and milled to course powder (10 mesh pass through).Further, four volumes of n-hexane was added to 100 gm of Trigonellafoenum-graecum seeds course powder and extracted at reflux temperature.n-Hexane fraction was filtered through Whatman filter no 1 and againthree times same extraction was carried out. After extraction, retentatewas dried at 80° C. for 5 h and then this was milled to obtain 40 meshpass through powder. In an alternative method, fat or oil from Fenugreekseeds were removed by Super Critical fluid extraction method usingliquid CO₂ as solvent. To increase the dietary fiber (Galactomannans)content, the enzymatic hydrolysis using Cellulase was carried out. Thegalactomannans content was determined by Megazyme kit (K-GALM 03/11) asper the manufacturer's instructions (Megazyme International Ireland,Bray Business Park, Bray, Co. Wicklow, IRELAND).

Lycium barbarum Seed Fibers

Goji, Goji berry or Wolfberry is the fruit of Lycium barbarum. The fruitof Lycium barbarum was dried and seeds were separated and milled tocourse powder. Further, four volumes of n-hexane was added to 100 gm ofLycium barbarum course powder and extracted at reflux temperature.n-Hexane fraction was filtered through Whatman filter no 1 and againthree times same extraction was carried out. After extraction, retentatewas dried at 80° C. for 5 h and then this was milled to obtain 60 meshpass through powder. Total dietary fiber was determined byEnzymatic-Gravimetric Method (AOAC 985.29).

Linum usitatissimum (Flax Seed) Fibers

Linum usitatissimum (also known as common flax or linseed or Flax) seedswere collected and milled to course powder (10 mesh pass through).Further, four volumes of n-hexane was added to 100 gm of Linumusitatissimum course powder and extracted at reflux temperature.n-Hexane fraction was filtered through Whatman filter no 1 and againthree times same extraction was carried out. After extraction, retentatewas dried at 80° C. for 5 h and then this was milled to obtain 40 meshpass through powder. Total dietary fiber was determined byEnzymatic-Gravimetric Method (AOAC 985.29).

Cocos nucifera Fibers

A matured coconut was procured from local market and dried. Further, theendosperm (coconut meat) was chopped to course and uniform sizematerial. Further, four volumes of n-hexane was added to 100 gm of Cocosnucifera course material and extracted at reflux temperature. n-Hexanefraction was filtered through Whatman filter no 1 and again three timessame extraction was carried out. After extraction, retentate was driedat 80° C. for 5 h and then this was milled to obtain 60 mesh passthrough powder. Total dietary fiber was determined byEnzymatic-Gravimetric Method (AOAC 985.29).

Zingiber officinale (ginger) rhizome fibers

Zingiber officinale rhizome, ginger root or simply ginger was dried andmilled to course powder (10 mesh pass through). Further, four volumes ofn-hexane was added to 100 gm of Zingiber officinale rhizome coursepowder and extracted at reflux temperature. n-Hexane fraction wasfiltered through Whatman filter no 1 and again three times sameextraction was carried out. In an alternative method, fat or oil fromFenugreek seeds were removed by Super Critical fluid extraction methodusing liquid CO₂ as solvent. After extraction, retentate was dried at80° C. for 5 h and then this was milled to obtain 40 mesh pass throughpowder. Total dietary fiber was determined by Enzymatic-GravimetricMethod (AOAC 985.29).

Emblica officinalis (Amla) fruit fibers

Emblica officinalis (Phyllanthus emblica), also known as Emblic, Emblicmyrobalan, Myrobalan, Indian gooseberry, Malacca tree, or Amla fromSanskrit Amalika. The fruit of Emblica officinalis was procured fromlocal market and dried, pulverized and passed through 60 mesh. Thispowder was used for the extraction of fibers. Total dietary fiber wasdetermined by Enzymatic-Gravimetric Method (AOAC 985.29).

Example 2—Acid Hydrolysis

Two grams of plant based natural fibers listed in Table 1 were dissolvedin 100 ml of HCl (0.10 M) and incubated at 37° C. with 100 rpm for 180min. Samples were taken at 0, 30, 60, 90, 120 and 180 min.Fructooligosaccharide (FOS; Tata Chemicals, India) was also taken in thestudy as reference to compare with natural fibers and starch (Potatosoluble starch; HiMedia, Mumbai, India) was also taken as control. Theincrease in reducing carbohydrates was measured by Dinitrosalicylatereagent (Nilsson and Bjorck 1988. Journal of Nutrition 118, 1482-1486).

TABLE 1 Total dietary fiber content of plant based natural fibersDietary Fibers (%, w/w) S. Soluble Insoluble No. Natural Fibers FibersFibers Total 1 Trigonella foenum- 65.42 ± 1.2  5.41 ± 0.4 75.24 ± 1.2graecum (Fenugreek) seed fibers 2 Lycium barbarum ND 38.05 ± 0.9 39.71 ±1.1 seed fibers 3 Linum usitatissimum 34.68 ± 0.4 12.73 ± 0.1 50.21 ±1.5 (Flax) seed fibers 4 Cocos nucifera 33.16 ± 0.7 36.16 ± 0.2 65.41 ±1.7 (Coconut) fibers 5 Zingiber officinale ND 41.55 ± 0.7 42.18 ± 0.8(Ginger) rhizome fibers 6 Emblica officinalis 10.15 ± 0.2 41.58 ± 0.552.07 ± 1.4 (Amla) fruit fibers Soluble Fraction 19.04 ± 0.5 1.527 ± 0.122.29 ± 0.4 Insoluble Fraction 27.05 ± 0.7  3.55 ± 0.1 33.03 ± 0.7 7Plantago ovata 51.13 ± 0.8 30.24 ± 0.8 83.24 ± 1.5 (Psyllium) Fibers 8Vaccinium oxycoccos 10.21 ± 0.6 39.81 ± 07  51.92 ± 1.3 (Cranberry) seedfibers ND, Not detected; Total dietary fiber was determined byEnzymatic-Gravimetric Method (AOAC 985.29).

Table 2 shows the effect of acid hydrolysis on (0.1 M HCl; 37° C., 100rpm) on Plant Based Natural Fibers. Total reducing sugar was determinedby Dinitrosalicylic acid (DNSA) method.

TABLE 2 Percentage of Total Reducing Sugar S. No. Plant Based NaturalFibers 0 min 30 min 60 min 90 min 120 min 180 min 1 Fenugreek seedfibers 3.79 ± 0.1 3.90 ± 0.1  3.95 ± 0.09 3.33 ± 0.1  4.22 ± 0.2  4.45 ±0.1 2 Lycium barbarum seed fibers 11.91 ± 0.2  12.05 ± 0.2  12.98 ± 0.3 11.94 ± 0.4  12.98 ± 0.3 12.51 ± 0.2 3 Flax seed fibers 1.24 ± 0.1  1.89± 0.09 1.97 ± 0.1 2.10 ± 0.1  1.98 ± 0.1  2.21 ± 0.1 4 Coconut Fiber 6.11 ± 0.09 6.51 ± 0.2 7.25 ± 0.1 8.35 ± 0.2  9.95 ± 0.2 10.05 ± 0.5 5Ginger rhizome fibers 4.02 ± 0.1 7.24 ± 0.5 7.98 ± 0.3  8.1 ± 0.7  7.8 ±0.2  7.5 ± 0.3 6 Amla Fruit Fiber 16.59 ± 0.2  16.14 ± 0.7  16.84 ± 0.3 15.16 ± 0.4  17.12 ± 0.6 16.98 ± 0.6 (Soluble + Insoluble) 7 AmlaSoluble Fiber 19.96 ± 0.3  24.75 ± 0.5  24.13 ± 0.6  23.42 ± 0.7  22.76± 0.8 23.17 ± 0.5 8 Amla Insoluble Fiber 6.98 ± 0.1 6.74 ± 0.1 6.92 ±0.2 6.95 ± 0.2  7.18 ± 0.1  7.60 ± 0.1 9 Psyllium husk Fiber  0.62 ±0.01  1.11 ± 0.09  1.28 ± 0.04 1.42 ± 0.1  1.51 ± 0.1  1.62 ± 0.1 10Cranberry seed Fiber 19.70 ± 0.4  19.15 ± 0.3  20.50 ± 0.9  19.76 ± 0.7 19.67 ± 0.9 20.60 ± 0.8 11 Fructooligosaccharide (FOS) 1.48 ± 0.1 6.29 ±0.1 8.96 ± 0.1 9.63 ± 0.2 10.47 ± 0.2 12.52 ± 0.4 12 Potato SolubleStarch 7.60 ± 0.2 21.43 ± 0.2  21.03 ± 0.5  25.05 ± 0.7  27.11 ± 0.734.20 ± 0.3

Example 3—Enzymatic Hydrolysis

100 mg of Pancreatin from Porcine pancreas 4×USP (Sigma-AldrichCorporation St. Louis Mo., USA) was dissolved in 100 ml of phosphatebuffer (50 mM; pH 7.0). Further, plant based natural fibers (2 gm) weredissolved in above Pancreatin solution and incubated at 37° C. with 100rpm for 180 min. Samples were taken at 0, 30, 60, 90, 120 and 180 min.FOS was also taken in the study as reference to compare with plant basednatural fibers and starch was also taken as control. The increase inreducing carbohydrates was measured with a Dinitrosalicylate reagent(Oku et al. 1984. Journal of Nutrition 114, 1574-1581). The effect ofenzymatic hydrolysis (0.1% Pancreatin in 20 mM PBS pH 7.0; 37° C., 100rpm) on plant based natural fibers is represented in Table 3. Totalreducing sugar was determined by Dinitrosalicylic acid (DNSA) method.

TABLE 3 Percentage of total reducing sugar S. No. Plant Based NaturalFibers 0 min 30 min 60 min 90 min 120 min 180 min 1 Fenugreek seedfibers 7.20 ± 0.1  8.15 ± 0.1 11.85 ± 0.8 11.35 ± 0.8 10.55 ± 0.1 10.70± 0.2 2 Lycium barbarum seed fibers 11.86 ± 0.2  18.11 ± 0.8 17.85 ± 0.217.75 ± 0.4 17.53 ± 0.8 18.21 ± 0.4 3 Flax seed fibers 1.12 ± 0.1  2.56± 0.05  2.57 ± 0.1  2.98 ± 0.1  2.78 ± 0.08  2.88 ± 0.04 4 Coconut Fiber8.85 ± 0.2 11.65 ± 0.2 14.25 ± 0.5 13.60 ± 0.2 10.90 ± 0.4  11.05 ± 0.085 Ginger rhizome fibers  4.14 ± 0.09  6.94 ± 0.2  7.12 ± 0.3  7.31 ± 0.4 6.81 ± 0.1  6.82 ± 0.09 6 Amla Fruit Fibers 16.05 ± 0.6  16.94 ± 0.216.48 ± 0.2 15.96 ± 0.2 17.40 ± 0.5 16.53 ± 0.7 (Soluble + Insoluble) 7Amla Soluble Fibers 20.10 ± 0.7  22.18 ± 0.8 20.51 ± 0.4 20.90. ± 0.8 23.72 ± 0.8 23.07 ± 0.5 8 Amla Insoluble Fibers 7.12 ± 0.1  7.58 ± 0.3 7.94 ± 0.6  8.19 ± 0.1  8.38 ± 0.01  8.45 ± 0.1 9 Psyllium husk Fiber1.05 ± 0.1  2.21 ± 0.1  2.26 ± 0.1  2.28 ± 0.02  2.29 ± 0.05  3.07 ± 0.110 Cranberry seed Fiber 19.74 ± 0.9  21.99 ± 0.8 24.33 ± 0.3 23.95 ± 0.423.58 ± 0.4 23.85 ± 0.7 11 Fructooligosaccharide (FOS)  1.10 ± 0.01 3.37 ± 0.09  3.12 ± 0.09  3.01 ± 0.1  3.43 ± 0.09  3.34 ± 0.08 12Potato Soluble Starch  7.45 ± 0.05 52.56 ± 0.9 54.06 ± 1.1 52.29 ± 1.252.10 ± 1.5 54.52 ± 1.1

Example 4—Growth Promotional Activity of Plant Based Natural Fiber withBacillus coagulans MTCC 5856

Single isolated colony of Bacillus coagulans MTCC 5856 was inoculatedinto MRS broth (pH 7.0±20; Himedia, Mumbai, India) and incubated at 37°C. with 120 rpm for overnight. Plant based natural fibers alone (0.5,1.0, 2.0%, w/v), and in MRS media (devoid of dextrose) (0.5, 1.0, 2.0%,w/v) were prepared. MRS broth and MRS (devoid of dextrose) were alsoprepared separately. Similarly, Fructooligosaccharide (FOS) was alsotaken in the study as reference control to compare with plant basednatural fibers. The final pH of all the media was adjusted to 7.0. Fivepercent of overnight grown Bacillus coagulans MTCC 5856 culture wasinoculated to all the flasks and incubated at 37° C. with 100 rpm for 24h. pH values at 0 h of incubation and after fermentation (24 h) werealso recorded. Samples were serially diluted in sterile saline and theviable count was enumerated by plating on glucose yeast extract agar(HiMedia, Mumbai, India) at 0 h and after fermentation (24 h). Theplates were incubated at 37° C. for 48 to 72 h. Each analysis wasperformed in triplicate at two different occasions. Average mean ofviable counts are expressed in log 10 cfu/ml (FIGS. 1A, 1B and 1C).

Example 5—Inhibition of E. coli Growth

The in-vitro experiment was designed to evaluate the effect of Plantbased natural fibers with probiotic bacteria Bacillus coagulans MTCC5856 for the inhibition of Gram negative pathogenic bacteria E. coli.Briefly, 2.0 g of plant based natural fibers were added to 100 ml ofdemineralized water. Psyllium husk Fiber and Flax seed fibers were added0.5 gm to 100 mL of demineralized water due to high gelling property.The pH was adjusted to 7.0±0.2 and autoclaved at 121° C. for 20 min.After sterilization, oxygen reducing enzyme Oxyrase (Oxyrase® for Broth,Oxyrase, Inc, Mansfield, Ohio, USA) was added to each flask. Bacilluscoagulans MTCC 5856 was grown on glucose yeast extract agar (Himedia,Mumbai, India) and E. coli ATCC 25922 was grown on trypticase soya agar(Himedia, Mumbai, India). Single isolated colony of both the cultureswas used and the turbidity of the bacterial suspension was adjusted to0.5 McFarland standards (equivalent to 1.5×108 colony forming units(CFU)/ml). One milliliter of E. coli ATCC 25922 was added to flaskcontaining plant based natural fiber. Similarly, in other group 1 ml ofE. coli ATCC 25922 and 1 ml of B. coagulans MTCC 5856 were added toflask containing plant based natural fiber. The flasks were incubated at37° C. with 100 rpm for 24 h. Samples were serially diluted in sterilesaline and the viable count of E. coli ATCC 25922 was enumerated byplating on Eosin Methylene Blue Agar (EMB Agar; HiMedia, Mumbai, India)at 0 h and after fermentation (24 h). The plates were incubated at 37°C. for 48 h. Each analysis was performed in triplicate at two differentoccasions. Average mean of viable counts are expressed in log₁₀ cfu/ml(FIG. 3).

Example 6—Production of SCFA by Bacillus coagulans MTCC 5856 Using PlantBased Natural Fibers

The in vitro fermentation with the Bacillus coagulans MTCC 5856 wascarried out by following method described by McBurney and Thompson(McBurney M I and Thompson L U. (1987) Effect of human faecal inoculumon in vitro fermentation variables. Brit J Nutr 58: 233-243) with somemodifications. Briefly, 2.0 g of glucose or Plant Based Natural Fiberswere added to 100 mL of demineralised water. Psyllium husk Fiber andFlax seed fibers were added 0.5 gm to 100 ml of demineralised water dueto high gelling property. The pH was adjusted to 7.0±0.2 and autoclavedat 121° C. for 20 min. After sterilization, oxygen reducing enzymeOxyrase (Oxyrase® for Broth, Oxyrase, Inc, Mansfield, Ohio, USA) wasadded to each flask, to induce anaerobic conditions. Five percent ofovernight grown Bacillus coagulans MTCC 5856 culture was inoculated toall the flasks and incubated at 37° C. with gentle shaken rpm for 24 h.The bottles were tightly closed and sealed with parafilm to maintainanaerobic conditions generated by the enzyme supplement. pH values at 0h of incubation and after fermentation (24 h) were also recorded. One mlof copper sulphate (10 g/L) was added to each sample to inhibit furthermicrobial growth (Sigma, St. Louis, Mo., USA). The analysis of shortchain fatty acids in the aforesaid fermentation samples was doneadopting the following parameters.

Reagents

1. Diethyl Ether (AR Grade)

2. H₂SO₄

3. RO Water

4. Sodium Chloride (AR Grade)

Chromatographic Conditions

Oven:

Rate Temperature Bold time Initial  80° C. 1.00 minute 8° C./minute 200°C. 2.00 minute

1. Post run temperature 220° C. 2. Post Time 5.0 min 3. Run time 18.00minInlet

Injection Volume 1 μl Temperature 250° C. Mode Split Split ratio 10:1Column

1. DB-FFAP (Terephthalic acid modified poly ethylene Glycol)

2. Dimensions: 30.00 m×250.00 mm×0.25 μm.

3. Carrier gas:Nitrogen

4. Flow: 1.0 ml/min

Detector

1. Type FID 2. Temperature 350° C. 3. Hydrogen Flow 30.0 ml/min 4. Airflow 300.0 ml/min 5. Make Up Flow 5.0 ml/min

Standard Solution Preparation

100.0 mg of each of Fatty acid standard (Acetic acid, Propionic acid andButyric acid) was weighed accurately in a 100 ml volumetric flask &dissolved in 50.0 mL of water and made up to the mark with water andmixed well (Stock solution). Further, 10.0 mL of the stock solution wasdiluted to 100.0 mL with water and mixed well to get standard solution.5 mL of standard solution was subjected to extraction as describedherein below.

1. Taken 5 ml of Standard solution/sample.

2. To Standard solution added 5 ml of water with vortexing for 0.5 min.

3. Adjusted pH to 1-1.5 with 3M H₂SO₄ with vortexing for 0.5 min.

4. Kept diethyl ether in −20° C. up to 1 hr before adding to thesample/Working Standard.

5. Added 10 ml of diethyl ether with vortexing for 1 min.

6. Added 4 g of Sodium Chloride with vortexing for 1 min.

7. Centrifuged to separate Water Layer & Diethyl Layer

8. Transferred 1.0 mL of Diethyl Ether layer in GC Vial & Injected.

Procedure:

1 μl each of extracted standard solution was injected into thechromatograph in triplicate and recorded the responses of major peaksdue to Acetic acid, Propionic acid and Butyric acid. The % RelativeStandard Deviation for area of peaks due to Acetic acid, Propionic acidand Butyric acid in triplet injections should not be more than 2.0%.Injected 1.0 μl each of extracted sample solution into thechromatograph. The content of Acetic acid, Propionic acid and Butyricacid was calculated as follows.

${{Content}\mspace{14mu}{of}\mspace{14mu}{individual}\mspace{14mu}{acid}\mspace{14mu}{in}\mspace{14mu}{ppm}} = \frac{{Sample}\mspace{14mu}{area} \times {{Std}.\mspace{14mu}{conc}.\mspace{14mu}({ppm})}}{{Standard}\mspace{14mu}{area}}$

The results of the chromatographic analysis are presented in FIG. 4 andrepresented herein below as Tables 4 and 5.

In Table 4 it may be noted that the production (mg/gram of fiber) ofshort chain fatty acid (Acetate, Propionate and Butyrate) was from plantbased natural fibers as a sole nutritional source in vitro batch-culturefermentation with B. coagulans MTCC 5856. FOS was used as referencecontrol in the study.In Table 5 it may be noted that the production (mg/gram if fiber) ofshort chain fatty acid (acetate, propionate and butyrate) was from plantbased natural fibers along with other nutrients in vitro batch-culturefermentation with B. coagulans MTCC 5856. In MRS media dextrose wasreplaced by plant based natural fibers for the production of SCFA. FOSwas used as reference control in the study. MRS media and Media devoidof Dextrose (MRSD) were also taken to compare for the production of thestudy.

TABLE 4 Short Chain Fatty Acids S. Plant Based Natural Fibers (mg/gramof Fibers) No. (alone) Acetate Butyrate Propionate 1 Fenugreek seedfibers 69.79 3.36 0.27 2 Lycium barbarum 77.18 6.56 0.24 seed fibers 3Flax seed fibers 109.5 5.93 0.27 4 Coconut Fibers 49.39 0.96 0.14 5Ginger rhizome fibers 1.62 0.20 42.35 6 Amla Fruit Fiber 3.87 0.30 79.99(Soluble + Insoluble) 7 Amla Soluble Fibers 5.50 0.23 76.55 8 AmlaInsoluble Fibers 1.05 0.25 44.10 9 Psyllium husk Fibers 1.70 0.14 38.1110 Cranberry seed Fibers 8.64 0.225 69.07 11 Fructooligosaccharide (FOS)1.07 0.07 21.61

TABLE 5 Plant Based Natural Fibers Short Chain Fatty Acids S. along withMRS Media devoid (mg/gram of Fibers) No. of dextrose Acetate PropionateButyrate 1 Fenugreek seed fibers 84.67 3.68 0.97 2 Lycium barbarum 61.055.41 0.75 seed fibers 3 Flax seed fibers 146.86 7.52 1.43 4 CoconutFibers 51.91 1.01 0.51 5 Ginger rhizome libers 118.72 7.90 1.32 6 AmlaFruit Fiber 94.11 6.46 1.06 (Soluble + Insoluble) 7 Amla Soluble Fibers90.99 9.37 1.24 8 Amla Insoluble Fibers 79.10 1.70 0.38 9 Psyllium huskFibers 53.24 3.23 0.56 10 Cranberry seed Fibers 110.89 9.58 1.69 11Fructooligosaccharide (FOS) 73.96 4.30 0.24 12 MRS Media 113.07 1.040.05 13 MRS Media devoid of 50.36 1.17 0.16 Dextrose (MRSD)

Example 7—Method of Preparing Turmeric Starch Fibers

Turmeric starch (brand name STARMERIC™-obtained from the spent materialresulting from the extraction of curcuminoids from the rhizomes ofCurcuma longa), was procured from the phytochemistry department of theSami Labs Limited [R&D arm of Sabinsa Corporation, N.J. USA] and wasused in the following experiments.

Example 8—Acid and Enzymatic Hydrolysis

The enzymatic hydrolysis procedure was carried out based on Oku et al.1984. Journal of Nutrition 114, 1574-1581). 100 mg of Pancreatin 4×USP(Sigma) was dissolved in 100 ml of phosphate buffer (50 mM; pH 7.0).Further, Turmeric starch (2 gm) was added to the above pancreatinsolution and incubated at 37±1° C. with 100 rpm for 180 min. Sampleswere taken at 0, 30, 60, 90, 120 and 180 min. FOS was also taken in thestudy as reference to compare with Turmeric starch and potato starch wastaken as positive control. The increase in reducing sugar was measuredwith a Dinitrosalicylate reagent.

The enzymatic hydrolysis was performed as per the procedure described inNilsson and Bjorck 1988. Journal of Nutrition 118, 1482-1486. TheTurmeric starch (2 gm) was added to 100 ml of HCl (0.10 M) and incubatedat 37±1° C. with 100 rpm for 180 min. Samples were taken at 0, 30, 60,90, 120 and 180 min. Fructooligosaccharide (FOS; Tata Chemicals, India)was also taken in the study as reference to compare with Turmeric starchand starch (Potato soluble starch: HiMedia, Mumbai, India) was taken aspositive control. The increase in reducing sugar was measured with aDinitrosalicylate reagent

Acid and enzymatic hydrolysis were performed for the turmeric starch todetermine its non-digestibility in human digestive system. An in-vitroexperiment was performed to mimic the human digestive system for theacid (HCl 0.1M, pH 1.4) and enzymatic hydrolysis (pancreatin containmixture of amylase, lipase and protease from porcine) of fiber. FOS wastaken as reference control for fiber and starch was taken as positivecontrol for its digestibility under in vitro condition.

In the acid and enzyme hydrolysis, potato soluble starch showed 14% and45% increase in total reducing sugar within 30 min and were found toremain in the range of 27% and 47% increase in total reducing sugar upto180 min respectively. Turmeric starch showed 18.0% increase in totalreducing sugar within 30 min and was found to remain in the range of19.66% increase in total reducing sugar up to 180 min when hydrolyzed bypancreatic enzymes (FIGS. 5 & 6).

The results suggest that turmeric starch was partially digested byenzymatic treatment. The total carbohydrate in the turmeric starch was58% and the initial total reducing sugar was 4.67%. After enzymatichydrolysis, there was an increase in total reducing sugar by 24.33%which resulted in 39.33% digestibility of turmeric starch. Therefore, itcan be concluded that turmeric starch contains about 60% ofenzymatically resistant starch. The in-vitro study suggested thatturmeric starch had non digestibility for acid and partial digestibilityto porcine pancreatic enzymes which could be used as a plant fiber forprebiotic application.

Example 9—Growth Promotional Activity of Turmeric Starch with Bacilluscoagulans MTCC 5856

A single isolated colony of Bacillus coagulans MTCC 5856 was inoculatedinto MRS broth (pH 6.0; Himedia) and incubated in shaker at 37±1° C.,120 rpm overnight. To evaluate the growth of Bacillus coagulans MTCC5856 using turmeric starch as carbon source, MRS media was used toevaluate the growth of Bacillus coagulans MTCC 5856 by replacing thedextrose with 2.5 g/L, 5 g/L, 7.5 g/L, 10 g/L of turmeric starch and 2.5g/L, 5 g/L, 10 g/L dextrose used as control in different set ofexperiments. MRS with Parameters like pH, OD (600 nm), Sporulation,Total viable count (TVC) was determined by serial dilution using GlucoseYeast Extract agar (Himedia) at 0 h and after fermentation (48 h). Totalcarbohydrate was quantified by Anthrone method. Viable count wasdetermined by pour plate method using GYE agar media. Turmeric starchwas found to be suitable for the growth of B. coagulans MTCC 5856 as thesole carbon source. However, FOS was slightly better than turmericstarch for enhancing the viable count of B. coagulans MTCC 5856 at 1.0and 2.0%, but at 0.5% concentration, B. coagulans MTCC 5856 viable countwas better compare to FOS (FIG. 7). Hence, turmeric starch could befurther explored as a prebiotic.

Example 10—Production of SCFA by Bacillus coagulans MTCC 5856 UsingTurmeric Starch

The in vitro fermentation of turmeric starch with the Bacillus coagulansMTCC 5856 was carried out and the short chain fatty acids were measuredby the method described in Example 6. The results of SCFA produced byBacillus coagulans MTCC 5856 in the presence of turmeric starch istabulated in Table 6.

TABLE 6 Production of Short Chain fatty acids by Bacillus coagulans MTCC5856 in presence of turmeric starch Short chain fatty acids (mg/g offibres) S. Time Acetic Butyric Propionic No. Samples (h) acid acid acid1 Turmeric Starch 24 442.3 10.2 3.8 (2.5 g/L) 48 664.0 8.6 0.02 72 628.06.5 0.6 2 Turmeric Starch 24 338.2 5.7 2.0 (5.0 g/L) 48 386.6 5.3 0.1 72593.4 2.8 0.3 3 Turmeric Starch 24 188.1 3.8 1.3 (7.5 g/L) 48 546.8 1.30.1 72 596.8 1.7 0.2 4 Turmeric Starch 24 182.7 7.0 0.8 (10.0 g/L) 48702.8 3.7 0.1 72 643.7 2.6 0.2 5 Dextrose 24 134.3 1.7 1.0 (10.0 g/L) 48428.0 5.5 1.2 72 472.1 1.4 1.0

Acetate was found to be the highest SCFA produced by Bacillus coagulansMTCC 5856 while fermenting turmeric starch and also dextrose. However,while fermenting starch at various concentrations; the production ofacetate was significantly higher than the dextrose group. Butyric acidand propionic acid production by Bacillus coagulans MTCC 5856 was alsoobserved to be relatively higher while fermenting turmeric starch, whencompared to the dextrose group. A concentration and time dependantproduction of SCFA by Bacillus coagulans MTCC 5856 was noticed whilefermenting turmeric starch. In conclusion, based on the in-vitrostudies, turmeric starch holds the potential of a prebiotic fiber.

While the invention has been described with reference to a preferredembodiment, it is to be clearly understood by those skilled in the artthat the invention is not limited thereto. Rather, the scope of theinvention is to be interpreted only in conjunction with the appendedclaims.

We claim:
 1. A method comprising co-culturing Bacillus coagulans MTCC5856 with turmeric starch.
 2. The method according to claim 1, whereinthe viable colony count of Bacillus coagulans MTCC 5856 is increased bythe co-culturing method.
 3. A method of producing short chain fattyacids, by co-culturing Bacillus coagulans MTCC 5856 with turmeric starchunder fermenting conditions to produce short chain fatty acids.
 4. Themethod according to claim 3, wherein the short chain fatty acids are oneor more selected from the group consisting of acetic acid, propionicacid, and butyric acid.
 5. The method according to claim 3, wherein theBacillus coagulans MTCC 5856 and the turmeric starch are fermented underanaerobic conditions.